ELISA | Enzyme linked immonosorbent assay | ELISA test | Types of ELISA | Direct and Indirect ELISA

Summary

This video introduces 'Bio Techniques Explained in Less Than Five Minutes,' focusing on the enzyme-linked immunosorbent assay (ELISA). ELISA is a diagnostic tool used to detect antigens or antibodies, vital for identifying viral diseases like HIV/AIDS. The video explains three ELISA variants: indirect, direct, and competitive, each serving different detection purposes. It illustrates how ELISA works, from setting up reactions in a 96-well plate to reading results through a colorimetric detector, providing qualitative and quantitative insights into antigen or antibody presence.

Takeaways

• 🔬 **ELISA Overview**: ELISA (Enzyme-Linked Immunosorbent Assay) is a colorimetric test used to identify substances like antigens or antibodies.
• 🌈 **Colorimetric Test**: ELISA uses color development reactions to provide qualitative and quantitative information about the presence of antigens or antibodies.
• 🧪 **Diagnostic Applications**: Widely used as a diagnostic test for many viral diseases, including HIV and AIDS.
• 🧫 **96-Well Plate Setup**: The reactions are set up in a 96-well plate and read using a colorimetric machine that measures absorbance at specific wavelengths.
• 📊 **Result Interpretation**: Results are displayed as absorbance versus concentration, indicating the quantity of the substance present.
• 🏥 **Indirect ELISA**: Used to detect antibodies against a known antigen, where the microtiter plate wells are coated with the antigen.
• 🩸 **HIV Detection Example**: Indirect ELISA can be used to detect HIV by identifying antibodies in a patient's serum against HIV viral antigens.
• 🔍 **Sensitivity and Specificity**: The presence or absence of color indicates whether the antibodies (or antigens) are present in the sample.
• 🧬 **Sandwich ELISA**: Used to detect the antigen of interest by using monoclonal antibodies to capture and detect the antigen.
• 📉 **Competitive ELISA**: Provides an understanding of how much antigen is present by competing with known antibodies for binding to the antigen.
• 📈 **Quantitative Analysis**: The intensity of color development in Competitive ELISA indicates the concentration of antigens in the sample.

Q & A

What is the primary purpose of the Enzyme-Linked Immunosorbent Assay (ELISA)?

ELISA is used to identify certain substances like antigens or antibodies. It provides both qualitative and quantitative information about the presence of these substances and is widely used as a diagnostic test for many viral diseases, including HIV and AIDS.

How does ELISA give qualitative and quantitative information about the presence of an antigen or antibody?

ELISA uses a colorimetric test that involves antibodies and a color development reaction. The reactions are set up in a 96-well plate and read by a spectrophotometer, which measures the absorbance at a specific wavelength. The result, displayed as absorbance versus concentration, provides insights into the quantity and presence of the substance.

What are the three variants of ELISA discussed in the script?

The three variants of ELISA discussed are Indirect ELISA, which detects antibodies against a known antigen; Sandwich ELISA, used to detect an antigen of interest; and Competitive ELISA, which helps understand the amount of antigen or antibody present in a sample.

How does Indirect ELISA work to detect antibodies?

In Indirect ELISA, wells of a microtiter plate are coated with a known antigen. The antibody to be tested is added, and if specific to the antigen, it binds to it. Then, an enzyme-linked secondary antibody is added, followed by a substrate that causes color development if the primary antibody is present, indicating the presence of the specific antibody in the sample.

What is the significance of using a 96-well plate in ELISA?

A 96-well plate is used in ELISA to facilitate multiple tests simultaneously, allowing for high-throughput screening. Each well can be coated with different antigens or antibodies, enabling the testing of various samples in a single assay.

Can you explain how Sandwich ELISA is used to detect the presence of an antigen?

In Sandwich ELISA, wells are coated with a monoclonal antibody that binds to the antigen of interest. After adding the patient's serum, another monoclonal antibody coupled with an enzyme is added. Upon adding the substrate, if the enzyme-coupled monoclonal antibody binds to the antigen, a color develops, indicating the presence of the antigen in the sample.

How does Competitive ELISA help in determining the quantity of antigen or antibody in a sample?

In Competitive ELISA, known antibodies are incubated with the antigen present in the sample. If the antigen concentration is high, most antibodies will bind to it, leaving few free to bind to the antigen-coated well surface, resulting in a faint color. Conversely, if the antigen concentration is low, more antibodies will be free to bind to the well surface, leading to a darker color, indicating a lower antigen concentration.

What is the role of the enzyme-linked secondary antibody in ELISA?

The enzyme-linked secondary antibody in ELISA binds to the primary antibody that has attached to the antigen. Once bound, it catalyzes a substrate to produce a colored product, which is then measured for absorbance to determine the presence and quantity of the specific antibody or antigen.

Why is color development in ELISA important?

Color development in ELISA is crucial as it provides a visual and measurable indication of the presence and quantity of the target substance. The intensity of the color correlates with the concentration of the antigen or antibody, allowing for both qualitative and quantitative analysis.

How does the absorbance reading in ELISA relate to the concentration of the substance being tested?

The absorbance reading in ELISA is directly proportional to the concentration of the substance being tested. A higher absorbance indicates a higher concentration of the antigen or antibody, while a lower absorbance suggests a lower concentration.